SuperFi-Cas9 exhibits extremely high fidelity but reduced activity in mammalian cells

Abstract

Several advances have been made to SpCas9, the most widely used CRISPR/Cas genome editing tool, to reduce its unwanted off-target effects. The most promising approach is the development of increased fidelity nuclease (IFN) variants of SpCas9, however, their fidelity has increased at the cost of reduced activity. SuperFi-Cas9 has been developed recently, and it has been described as a next-generation high fidelity SpCas9 variant, free from the drawbacks of the first-generation IFNs. In this study, we characterized the on-target activity and the off-target propensity of SuperFi-Cas9 in mammalian cells comparing it to first-generation IFNs. SuperFi-Cas9 demonstrated strongly reduced activity but exceptionally high fidelity exhibiting features that are in many aspects similar to those of the first-generation variants, such as evo- and HeFSpCas9. When combined with ABE8e, SuperFi-Cas9 produced DNA editing with high activity rate as well as high specificity by reducing both bystander and SpCas9-dependent off-target base editing.

Comparative analysis of CRISPR off-target activity discovery tools followingex vivoediting of CD34+hematopoietic stem and progenitor cells

“…Since HiFi Cas9 16 reduces editing at OT sites without compromising on-target editing, it is also likely that the risk of translocations/inversions between the on-target and OT sites are reduced accordingly and has been shown in one example 19 . Furthermore, other high-fidelity Cas9 enzymes as well as gRNA innovations have been reported 17,39,40 , and continued efforts attempt to increase activity and specificity of Cas9 via protein engineering. This may be especially important as genome editing technology begins to be adapted for in vivo delivery 7 .…”

“…Chemically modified gRNAs used to edit HSPCs were purchased from Synthego (Menlo Park, CA, USA). The gRNA modifications added were the 2′-O-methyl-3′-phosphorothioate at the three terminal nucleotides of the 5′ and 3′ ends 39 . All Cas9 protein (both Alt-R ® S.p.…”

Identification and Analysis of Small Molecule Inhibitors of CRISPR-Cas9 in Human Cells

“…We surmise that a cocktail of small-molecule compounds with different structures may be more effective at inhibiting the Cas9 nuclease. In a recent study, SpCas9 variants that could selectively cleave target sites were engineered using a crystal-structure-guided design [ 49 ], but the variant is less active in mammalian cells than the wild-type SpCas9 [ 50 ]. The mutation sites of the variants are concentrated in the region where SpCas9 interacts with DNA distal from the protospacer adjacent motifs (PAM).…”

Recent Advances in Improving Gene-Editing Specificity through CRISPR–Cas9 Nuclease Engineering

“…The competition assay showed that SuperFi-Cas9 displayed a 6.3-fold preference for on-target DNA relative to DNA bearing 18–20 mismatches, while WT SpCas9 showed only a low preference ratio of 1.55 fold [ 28 ]; this result indicated that SuperFi-Cas9 possessed a good ability to discriminate between on- and off-target substrates. A recent study (in the preprinted form) from an independent lab showed that SupperFi-Cas9 showed high-fidelity but significantly reduced on-target activity in mammalian cells [ 72 ].…”

“…Yeast cells [19], 293multiEGFP, 293blastEGFP, and HEK293T cells [19,29,98] SpartaCas T cells [24] LZ3 Cas9 HEK293T cells [29], K562 cells [29], U2OS cells [29] miCas9 induced pluripotent stem cells [32], airway epithelial cells [32], fibroblast cells [32], Jurkat cells [32], Ad293 cells [32] SuperFi-Cas9 HEK293 cells [72], neuro-2a mouse neuroblastoma cells [72] eSaCas9 HEK293 and HEK293T cells [18,22], human retinal pigmented epithelium cells [22] SaCas9-HF HEK293 and HEK293T cells [22,106], human retinal pigmented epithelium cells [22] efSaCas9 HEK293 cells [17], HeLa cells [17], HT-1080 cells [17] SaCas9-Q414A HEK293 cells [17], HeLa cells [17], HT-1080 cells [17] KKH-SaCas9-SAV1 HEK293T cells [31], SK-N-MC cells [31], MHCC97L cells [31], OVCAR8-ADR cells [31] KKH-SaCas9-SAV2 HEK293T cells [31], SK-N-MC cells [31], MHCC97L cells [31], OVCAR8-ADR cells [31] These engineered Cas9 variants have reduced off-target effects while maintaining the cleavage activity to different extents. Thus, choosing suitable Cas9 proteins or variants is critical for a specific gene-editing task.…”